Methods We generated murine CD103+ cDC1s in vitro and examined their expression of cDC1-related factors, antigen cross-presentation activity, and accumulation in tumor-draining lymph nodes (TdLNs). The antitumor efficacy of the in vitro-generated CD103+ cDC1s was studied in murine melanoma and osteosarcoma models. We evaluated tumor responses on vaccination with CD103+ cDC1s, compared these to vaccination with monocyte-derived DCs (MoDCs), tested CD103+ cDC1 vaccination with checkpoint blockade, and examined the antimetastatic activity of CD103+ cDC1s.

Conclusions Our data indicate an in vitro-generated CD103+ cDC1 vaccine elicits systemic and long-lasting tumor-specific T cell-mediated cytotoxicity, which restrains primary and metastatic tumor growth. The CD103+ cDC1 vaccine was superior to MoDCs and enhanced response to immune checkpoint blockade. These results indicate the potential for new immunotherapies based on use of cDC1s alone or in combination with checkpoint blockade.

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Background

T cell-based immunotherapy and antibody-mediated immune checkpoint blockade are among the most exciting advances in cancer therapy over the past decade, eliciting durable control of several cancers and prolonging survival rates.1 2 Nonetheless, limitations exist with current immunotherapies including non-responsiveness or adverse events.3 Thus, approaches to improve the specificity, effectiveness, and safety of cancer immunotherapy across patient populations and cancer types are needed.

Dendritic cells (DCs) are the principal antigen-presenting cells of the immune system and therefore shape adaptive, antitumor immunity.4 These features indicate DCs as a promising tool for anticancer treatment.5–7 The majority of DCs used in clinical trials have been generated from human CD14+ monocytes (MoDCs) or CD34+ progenitors in culture.8 While these DCs can be produced in abundance and are capable of inducing tumor-specific T cells with minimal side effects, their efficacy remains limited.7–9 More recently, specific DC populations including plasmacytoid DCs (pDCs) and type 2 conventional DCs (cDC2s) have yielded clinical responses,10 11 yet these subsets are relatively sparse in vivo. The efficacy or feasibility of current DC vaccines, therefore, may be limited by issues such as use of suboptimal or rare DC subsets.

Type 1 cDCs (cDC1s) exhibit several features that predict important roles in activating antitumor immunity, and abundance of cDC1s within tumors correlates with improved patient outcomes and response to immune checkpoint blockade.12 13 The cDC1 subset possesses antigen uptake, antigen presentation, and antigen cross-presentation abilities. Moreover, migratory CD103+ cDC1s transport tissue or tumor antigens to lymph nodes (LNs) and elicit antigen-specific CD8+ T cell responses.14–18 CD103+ cDC1s can be recruited to tumors by T cell-expressed chemokines including XCL1, where they participate in further T cell recruitment through expression of chemoattractants such as CXCL10.12 19 Consistent with these functions, lymphoid organ-resident CD8α+ cDC1s induced CD8+ T cell responses and protected mice against melanoma engraftment, while treatments to expand and activate locally recruited CD103+ cDC1s increased the efficacy of B-raf kinase (BRAF) inhibition and PD-1 blockade in controlling melanoma.18 20 Collectively, these features suggest cDC1-based vaccines will elicit antitumor activity, yet this concept requires further validation. Moreover, whether cDC1-based vaccines protect from metastatic disease is important to examine, as metastasis is a primary cause of mortality in patients with cancer.

Melanoma and melanoma metastatic disease are responsive to immunotherapies such as checkpoint blockade.2 7 A number of other tumor types, however, remain poorly responsive or refractory. In particular, pediatric solid tumors are frequently non-responsive to immunotherapy. Additionally, these tumors often develop resistance to standard treatments, leaving few clinical options and a need to identify novel approaches for young patients with cancer.

Osteosarcoma is the most common primary malignancy of bone affecting pediatric and adult patients. Chemotherapy and surgery are standard treatments, yet the 5-year survival rate is <20% for osteosarcoma patients who present with metastases or relapse following treatment. Negligible improvements have occurred in osteosarcoma therapeutic options over the past 25 years.21 22 Mifamurtide, a liposome-encapsulated immunotherapy that activates pulmonary macrophages, improved the disease-free and overall survival of patients with osteosarcoma lung metastases.23 By contrast, MoDC vaccines have yielded little to no clinical responsiveness,24–26 while checkpoint blockade with PD-1 or CTLA-4 antibodies led to objective clinical response rates of approximately 5% in osteosarcoma patients.27 28 These results underscore the critical need to improve therapeutic options for osteosarcoma.

Here, we tested the efficacy of in vitro-generated CD103+ cDC1s in preclinical murine tumor models, using the well-established B16 melanoma as well as the K7M3 osteosarcoma model. We found administration of poly I:C-activated, tumor antigen-loaded CD103+ cDC1s suppressed primary melanoma and osteosarcoma growth, elicited a systemic effect to control untreated bilateral tumors, and restrained distal lung metastasis. These responses were associated with an increase in interferon-γ (IFN-γ)+ CD8+ T cells and tumor antigen-specific CD8+ T cells. Our data suggest an in vitro-generated cDC1-based vaccine offers a novel strategy for cancer treatment including tumors that are refractory to other therapeutic options.

To evaluate potential mechanisms for the improved response to CD103+ cDC1 vaccination, we compared DC amounts in vaccinated and unvaccinated tumors and corresponding TdLNs 40 hours following i.t. delivery. Congenic CD45.1+ CD103+ cDC1s or CD45.1+ MoDCs were used in these assays to distinguish from endogenous populations. CD45.1+ CD103+ cDC1s were detected in tumors and TdLNs on the side of i.t. injection only (figure 2D–F). In addition, CD45.1+ CD103+ cDC1s were more abundant in TdLNs compared with endogenous cells, as judged by relative frequencies of CD45.1+ versus CD45.2+ CD103+ cDC1s on the treatment side (figure 2F). By contrast, the majority of CD45.1+ MoDCs remained within injected tumors; CD45.1+ MoDCs were rarely observed in TdLNs (figure 2G–I). The chemokine receptor CCR7, which mediates DC migration to LNs, was expressed at higher amounts on tumor-associated CD45.1+ CD103+ cDC1s compared with CD45.1+ MoDCs, while TdLN-associated populations expressed similar CCR7 amounts (online supplementary figure S2D). In addition, despite i.t. injection of equivalent DC numbers, CD45.1+ MoDC amounts were approximately 10% of CD45.1+ CD103+ cDC1s within tumors (figure 2D,G). These results suggest CD103+ cDC1s persist in tumors longer, express greater amounts of CCR7, and have superior TdLN accumulation versus MoDCs on vaccination.

We analyzed DC proliferation and survival in vaccine conditions, to further understand the improved efficacy of CD103+ cDC1s. These assays revealed modest but non-significant differences in Ki67 status prior to vaccination, and no detectable differences 40 hours following i.t. delivery, suggesting similar proliferation rates (figure 2J). By contrast, the activated CD103+ cDC1 vaccine contained fewer apoptotic and dead cells, and a greater proportion of viable cells prior to i.t. delivery, relative to the MoDC vaccine (figure 2K). In addition, CD103+ cDC1s showed enhanced viability following delivery to melanoma tumors versus MoDCs, although the viability of both populations 40 hours after vaccination was low (figure 2K and online supplementary figure S2E). These results indicate CD103+ cDC1s have improved survival following antigen and TLR agonist stimulation, as well as on exposure to melanoma tumors, compared with MoDCs, suggesting enhanced CD103+ cDC1 viability contributes to the efficacy of this population as a tumor vaccine.

Immune responses elicited by CD103+ cDC1 and MoDC vaccines in melanoma. Mice bearing bilateral B16-OVA tumors were vaccinated on one side with CD103+ cDC1s, MoDCs, or PBS as described in the legend to figure 2. (A–C) The number of tumor-infiltrating CD8+ T cells (A), and numbers and proportions of OVA-specific CD8+ T cells in tumors (B, C) were determined 14 days following vaccination in tumors that received the DC vaccine (treated, (T)) and in distal untreated (NT) tumors, as indicated. (D) The ability of vaccine-derived CD45.1+ CD103+ cDC1s or CD45.1+ MoDCs, purified 40 hours following i.t. delivery, to stimulate OT-I CD8+ T cell proliferation was determined in coculture assays in vitro. Proliferating OT-I CD8+ T cells were measured at 72 hours; assays were performed in the presence of 20 ng/mL GM-CSF. (E–G) The numbers of CD4+ T cells (E), CD4+ CD25+ Foxp3+ Treg (F), and CD4+ Foxp3- effector T cells (Teff) (G) were determined 14 days following vaccination in tumors that received the DC vaccine (treated, (T)) and in distal untreated (NT) tumors, as indicated. (H) The percentage of Tregs within the total CD4+ T cell population in tumors from mice vaccinated as indicated, 14 days following vaccination. (I) The ability of vaccine-derived CD45.1+ CD103+ cDC1s or CD45.1+ MoDCs, purified from tumors 40 hours following i.t. delivery, to stimulate OT-II CD4+ T cell proliferation was determined in coculture assays in vitro. Proliferating OT-II CD4+ T cells were measured at 72 hours; assays were performed in the presence of 20 ng/mL GM-CSF. (A, B, D–I) Data shown as mean±SEM. (A–I) Representative (C) or cumulative (A, B, D–I) results from two independent experiments; n=9 (PBS), n=5 (MoDC), n=4 (CD103+ cDC1). Results were analysed by one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. ANOVA, analysis of variance; cDC1, type 1 conventional dendritic cells; i.t., intratumoral; MoDC, monocyte-derived dendritic cell; OVA, ovalbumin.

The CD103+ cDC1 vaccine also promoted an increase in tumor-infiltrating CD4+ T cells, including Treg and T effector subsets, in vaccinated tumors but not distal untreated tumors, compared with MoDC vaccination or PBS treatment (figure 3E–G). The relative frequency of Tregs within the tumor-infiltrating CD4+ T cell population was reduced in DC vaccine-treated mice, however, compared with controls (figure 3H). Furthermore, vaccine-origin CD45.1+ CD103+ cDC1s and CD45.1+ MoDCs isolated from tumors were equivalently effective at stimulating CD4+ T cell proliferation in vitro, although CD4+ T cell proliferation was notably reduced compared with CD8+ T cell proliferation in these conditions (figure 3D,I). Tumor-infiltrating B cells, monocytes, macrophages, cDC1s, and cDC2s were not significantly different in treated or untreated tumors among the three groups, while neutrophils were induced in tumors on CD103+ cDC1 but not MoDC vaccination (online supplementary figure S3B).

To examine whether individual or combination immunotherapies induced long-lasting antitumor immunity, we rechallenged the surviving mice from each therapeutic group with K7M3 tumors. Tumor-free survival was observed in the majority of mice up to 370 days following tumor rechallenge (figure 6D). These data indicate single or combination immune therapy is capable of establishing immunological memory responses against murine K7M3 osteosarcoma, with the most potent response mediated by combining CD103+ cDC1 vaccination and CTLA-4 blockade.

Discussion

The cDC1 subset mediates antitumor immune responses in mice and is associated with improved outcomes in human cancer.6 12–14 Nonlymphoid organ cDC1s (ie, CD103+ cDC1s in mice or CD141+ DCs in humans) infiltrate solid tumors and transport tumor antigens to LNs to induce tumor immunity.4 15–18 Thus, we employed a previously described culture system29 to produce large numbers of CD103+ cDC1s for use as a cancer vaccine. Our data indicate in vitro-generated CD103+ cDC1s produce T cell activating cytokines and chemokines, cross-present exogenous antigen to naïve CD8+ T cells to induce their proliferation, and migrate to TdLNs in vivo. Significantly, in vitro-generated CD103+ cDC1s control primary and metastatic melanoma and osteosarcoma tumors on vaccination. The CD103+ cDC1 vaccine also shows improved activity over a MoDC vaccine, and enhances the response of osteosarcoma to checkpoint blockade. These results support the idea that functional CD103+ cDC1s can be generated in abundance in cultures to provide an effective form of immunotherapy.

MoDC-based vaccines have been employed for years, as these cells can be expanded efficiently in culture. Furthermore, MoDC vaccines are relatively safe and can induce antitumor immunity; however, their clinical efficacy is limited.36 Hence, recent efforts have focused on evaluating distinct DC types in cancer immunotherapy.37 38 Vaccines based on pDC or cDC2 subsets have shown some promise.10 11 Moreover, purified murine lymphoid organ cDC1s demonstrated effective tumor control.20 The number of naturally occurring DCs that can be harvested for clinical use is limited, however, posing challenges for effective translation.39 40 In mice, this hurdle was overcome by generating abundant amounts of CD103+ cDC1s in culture29; BM cells from one mouse can generate sufficient quantities of CD103+ cDC1s for vaccination of 30–50 tumor-bearing animals. Importantly, murine and human DC populations share developmental and functional traits, including similar transcriptional networks, cytokine responses, and T cell-activating abilities.31 41 These data suggest culture systems that effectively expand human cDC1s may be feasible; for instance, optimizing use of Flt3L in cultures with blood-derived DC progenitors to generate human CD141+ DCs, and testing the ability of this population in tumor vaccine strategies.

The activity of current DC vaccines may be affected by DC survival rates, persistence in tumors, and migratory activity to TdLNs. We found CD103+ cDC1s remain longer in melanoma tumors, and accumulate in TdLNs more efficiently than MoDCs, as judged by their relative abundance 40 hours after vaccination. Moreover, CD103+ cDC1s showed prolonged survival following antigen stimulation, TLR agonist treatment, and exposure to the melanoma environment versus MoDCs. The delivery of tumor antigens to TdLNs is critical for effective induction of antitumor immunity by DCs.42 Taken together, these results suggest functional traits exhibited by CD103+ cDC1s in the tumor environment contribute to their superior efficacy as a tumor vaccine.

The MoDC vaccine preferentially induced IL-4-producing CD4+ (Th2) T cells in TdLN, and MoDCs isolated from TdLNs were significantly more effective in stimulating OT-II CD4+ T cell proliferation in vitro versus CD103+ cDC1s. Moreover, MoDC vaccination was generally associated with increases in T cell amounts in TdLNs relative to CD103+ cDC1 vaccination. At first glance, the latter data seem inconsistent with the superior efficacy of the CD103+ cDC1 vaccine. Importantly, however, MoDC vaccination did not significantly expand tumor-infiltrating T cell populations, and MoDCs isolated from tumors were inferior to CD103+ cDC1s in eliciting CD8+ T cell proliferation in vitro. MoDC vaccination also failed to induce tumor antigen-specific T cell responses to an appreciable amount in vivo. Collectively, these results indicate the CD103+ cDC1 vaccine is significantly more potent at stimulating antitumor immune responses relative to MoDCs. Furthermore, as Th2 cells produce cytokines such as IL-10, IL-4, and IL-5, which limit cytotoxic T cell proliferation and drive macrophages to an M2 phenotype,43 our data suggest MoDCs have potential to promote an immune suppressive environment. Additional work to elucidate underlying immune mechanisms activated by each vaccine, such as determination of tumor macrophage phenotypes and cytokine profiles, is necessary to fully understand and improve DC-mediated vaccine responses.

Our work and studies by others suggest that multiple routes of cDC1 vaccine delivery are effective. A prior study used intradermal delivery of lymphoid organ CD8α+ cDC1s loaded with dead tumor cell-derived antigens,20 while we employed i.t. as well as i.v. delivery mechanisms. The i.t. delivery route was expected to enable CD103+ cDC1 migration to TdLNs and subsequent activation of antitumor immune responses; however, it was unclear a priori whether administration of CD103+ cDC1s i.v. would lead to effective antitumor immunity since cDC1s terminally differentiate in lymphoid organs or tissues.40 41 As delivery of poly I:C-stimulated and tumor antigen-loaded CD103+ cDC1s i.v. suppressed experimental lung metastases, the results suggest this route supports CD103+ cDC1 survival, antigen presentation, and elicitation of durable T cell responses. Thus, i.v. delivery mechanisms may be effective for DC vaccines that use tissue-resident cDC1s, therefore facilitating DC-based vaccine approaches for disseminated or metastatic tumors.

Although checkpoint blockade is a current treatment for many cancers including melanoma, osteosarcoma has been largely refractory to immunotherapy with the exception of mifamurtide.22 44 Mifamurtide is also effective in melanoma,45 suggesting melanoma and osteosarcoma share features of therapeutic responsiveness. We examined the effect of CD103+ cDC1 vaccination on primary and metastatic osteosarcoma tumor growth, as well as the efficacy of combination treatment with checkpoint blockade. Significantly, combination of CD103+ cDC1 vaccination and CTLA-4 blockade led to osteosarcoma tumor regression in all mice. Moreover, this combination treatment appeared to induce long-term immune memory, as animals rechallenged with osteosarcoma did not develop tumors. Combination treatment with CD103+ cDC1s and PD-1 blockade was less effective, by contrast. CTLA-4 and PD-1 have distinct roles in limiting T cell priming or effector function, respectively.46 47 Hence, we expect CD103+ cDC1 vaccination operates in part by priming naïve T cells, particularly CD8+ T cells, and this function may be enhanced by combination treatment with CTLA-4 blockade. Further studies are required to delineate the immunological mechanisms by which CD103+ cDC1 vaccination and CTLA-4 blockade elicit anti-tumor immunity; however, these results suggest the exciting potential for combination immunotherapy in the treatment of osteosarcoma.

Conclusions

Our findings reveal potent systemic and anti-metastatic efficacy of an in vitro-generated CD103+ cDC1 vaccine. CD103+ cDC1 vaccination elicits IFN-γ-producing CD8+ T cell and Th1 responses, as well as long-lasting tumor antigen-specific T cell activity. CD103+ cDC1 vaccination was superior to MoDC vaccination, and enhanced response to checkpoint blockade therapy in osteosarcoma, a tumor type that is refractory to the majority of current immunotherapies. Our data suggest the potential for novel cellular immunotherapies based on use of cDC1s alone or in combination with checkpoint blockade.

Acknowledgments

We thank Kathryn Newton and Dr. Yuanzheng Yang for technical assistance, Dr. Bhakti Patel for discussion and review of the manuscript, Drs. Gabriela Raso and Ximing Tang for evaluating osteosarcoma pulmonary metastasis, and the MD Anderson South Campus Flow Core for experimental advice and assistance.

. Liposomal muramyl tripeptide (CGP 19835A lipid) therapy for resectable melanoma in patients who were at high risk for relapse: an update. Cancer Biother Radiopharm1998;13:363–8.doi:10.1089/cbr.1998.13.363

Footnotes

Contributors YZ conceptualized the studies, designed and performed experiments, collected and analyzed data, and wrote the manuscript. NS and ESK conceptualized studies and designed experiments. NS, TTC, OK, RLB, YBM and HSL developed methodology, performed experiments and collected and analyzed data. SSW conceptualized the studies, designed experiments, wrote the manuscript, contributed to funding acquisition, administered the project, and supervised the study. All authors read, edited and approved the final manuscript.

Funding This work was supported by grants from the NIH NIAID (R01AI109294 and 3R01AI109294-04S1 to SSW), the MD Anderson Center for Inflammation and Cancer (to SSW and HSL), a Research Training Award from the Cancer Prevention and Research Institute of Texas (CPRIT RP170067 to TTC, OK and RLB), and the MD Anderson NIH NCI P30CA016672 grant (supporting the MD Anderson South Campus Flow Core).

Data availability statement All data relevant to the study are included in the article or uploaded as online supplementary information. Additional information regarding data may be obtained from the authors on reasonable request.

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